1. When a dimer is refolded with equal amounts of labeled and unlabeled protein (or
differently labeled proteins), one gets 25% fully labeled dimers, 25% fully
unlabeled dimers, and 50% asymmetrically labeled dimers. So, in your case, the
signals you would expect from the doubly labeled protein should be from 25% of
total protein (the fraction that has one monomer with 15N and the other monomer
with 13C). So, the S/N will not be as high as you might otherwise expect.
2. Do you know if you have really have 8mg/500 uL total protein concentration? How
did you verify the concentration? I ask this because many proteins tend to crap out
to some extent or the other during refolding.
To make sure loss of protein during refolding is not the problem, one thing to try
- if you haven't already- is to do the refolding on unlabeled molecules. Check
soluble protein concentration and NMR spectra before and after refolding (simple 1D
NOE) to prove to yourself the % recovery after folding.
Hope this helps,
Sudha Veeraraghavan
Yu-Feng Tong wrote:
> Hello all,
>
> I'd like to know anyone had the experience on carrying out 4D
> 13C,15N-separated HMQC-NOESY-HSQC experiment and/or the experience on
> solving the structure of dimer. Here is my puzzle:
>
> I'm trying to determine the interfacial NOE of a dimer ( 97 a.a.
> monomer, m.w. ~11,000). I have prepared an 8mg/500uL equal molar ratio
> 15N/13C asymmetric labelled sample (4mg 15N labelled, 4mg 13C labelled,
> mixed and denatured by 6M GdmHCl, incubated at 4 degree overnight, then
> dialysized to renature the protein), then I carry out the 1D version of
> a 4D 13C,15N HMQC-NOESY-HSQC (Muhandiram, Xu, Kay: J. Biomol. NMR 3,
> 463-70 (1993)), i.e. acquire the first fid of a 4D experiment.
>
> To my surprise, the s/n ratio is very low even I accumulate 2048
> transients. Actually the s/n is 2 after 2k scans. For a comparison, I
> found the s/n of a 15N,13C double labelled sample (8mg/500uL too,
> identical acquisition parameters) to be 31 after 32 scans for a 1D
> version HMQC-NOESY-HSQC.
>
> I have suspected the protein to be a monomer, but size exclusion
> chromatography data show the protein to be larger than a 150 a.a.
> protein. And I also found Tracy M. Handel had used a ns=2048 for 2D
> version of the 4D experiment to identify interfacial NOE for MCP-1
> protein, (yet ns=64 for a 3D version)
>
> Any hint is greatly appreciated.
>
> Yu-Feng
>
> --
> Yu-Feng Tong||ͯÓî·å
> NMR Group, Dept. Enzymology
> Inst. Biophysics, Academia Sinica
> 15 Datun Road, Beijing, P.R.China, 100101
> _______________________________________________________________________________
> Life is an everlasting game of Weiqi(igo, baduk).
--
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Dr. Sudha Veeraraghavan
Asst. Prof
Structural Biology Research Center, MSB 6.130
Dept Biochem & Mol Biol
UT-Houston Medical School
6431 Fannin
Houston, TX 77030
Tel : 713-500-6089
Fax : 713-500-0651
http://www-bmb.med.uth.tmc.edu/programfac/Veeraraghavan/Veeraraghavan.html
Received on Tue Mar 05 2002 - 18:41:47 MST